Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins Determination of regulated mycotoxins and enniatins and beauvericin in cereals Carlos Oliveira Gonçalves Elena Cubero-Leon Vytautas Tamosiunas Carsten Mischke Stefanka Bratinova Joerg Stroka 2017 EUR 28790 EN
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Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins
Determination of regulated
mycotoxins and enniatins
and beauvericin in cereals Carlos Oliveira Gonçalves Elena Cubero-Leon Vytautas Tamosiunas Carsten Mischke Stefanka Bratinova Joerg Stroka
2017
EUR 28790 EN
This publication is a Technical report by the Joint Research Centre (JRC), the European Commission’s science and knowledge service. It aims to provide evidence-based scientific support to the European policymaking process. The scientific output expressed does not imply a policy position of the European Commission. Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use that might be made of this publication. Contact information
Name: Joerg Stroka Address: European Commission, Joint Research Centre Directorate F - Health, Consumers & Reference Materials, Retieseweg 111, 2440 Geel, Belgium Email: [email protected] Tel.: +32 14 571229 JRC Science Hub
https://ec.europa.eu/jrc JRC108384 EUR 28790 EN
PDF: ISBN 978-92-79-73717-6 ISSN 1831-9424 doi:10.2760/52884
The reuse of the document is authorised, provided the source is acknowledged and the original meaning or message of the texts are not distorted. The European Commission shall not be held liable for any consequences stemming from its reuse. How to cite this report: Author(s), Carlos Oliveira Gonçalves, Elena Cubero-Leon, Vytautas Tamosiunas, Carsten Mischke, Stefanka Bratinova, Joerg Stroka, Report on the 2016 Proficiency Test of the European Union
Reference Laboratory for Mycotoxins: determination of regulated mycotoxins and enniatins and beauvericin in
Annex 7. Kernel density plots ............................................................................ 42
Annex 8. Distribution of individual results and respective uncertainties (k=2) ......... 46
Annex 9. Method conditions, quantification approaches and quality control ............. 48
Annex 10. Method validation data ...................................................................... 63
1
Executive summary
The number of known mycotoxins, their precursors and metabolites has been steadily increasing over the past years. The European Commission puts special emphasis on the need to monitor the co-occurrence of mycotoxins of various families at levels that allow for a sound risk assessment, taking into account possible additive or synergistic effects. Prior any regulatory action is taken for mycotoxins for which a health concern has been expressed (e.g. enniatins and beauvericin) valid data on their prevalence in food is required.
LC-MS-based multi-mycotoxin methods have the potential of streamlining and widening the monitoring work carried out by the official control laboratories. Although many practical advantages have been recognised, such methodologies have not been adopted by all routine laboratories and only a handful of such methods are just on the verge to become standardised. It is of great interest to assess how well laboratories using diverse sample preparation methodologies and determination techniques perform.
Therefore, a proficiency test was organised by the European Union Reference Laboratory (EURL) for Mycotoxins for this purpose. The focus was the assessment of the measurement performance of EU Member States laboratories regarding the determination of aflatoxin B1, deoxynivalenol, zearalenone, fumonisins B1 & B2,T-2 & HT-2 toxins, enniatins B, B1, A, A1 and beauvericin in two test materials (corn and oat) using single- or multi-mycotoxin methodologies.
Fifty-three laboratories, among them thirty-six National Reference Laboratories for mycotoxins in food and feed from the 28 EU Member States and 17 Official Control Laboratories, participated in the PT. For the regulated mycotoxins, 83.7 % of the results were rated with satisfactory z-scores. The performance of the laboratories was best for AFB1 (94 %), followed by DON (91 %), ZON (89 %), FB1 (87 %), FB2 (78 %), T-2 (75 %) and HT-2 (64 %). Additionally, 11 laboratories submitted results for all enniatins and beauvericin. LC-MS/MS is gaining much preference as it allowed for the determination of all the proposed analytes (12) in the test materials. Nevertheless, the results provided by multi-mycotoxin methodologies did not differ statistically from those produced by single-analyte procedures. Many participants uphold the will to implement a methodology to analyse enniatins and beauvericin in the near future, while other laboratories' methods require improvements in the extraction efficiency and sensitivity.
2
Acknowledgements
The organisers of the study would like to thank the colleagues involved in the project for their support. The laboratories that participated in this exercise, listed in Table 1, are also immensely acknowledged.
Table 1. Participating laboratories Department Country
LVA GmbH Austria AGES GmbH Austria CODA-CERVA Belgium Bulgarian Food Safety Agency Bulgaria Andrija Stampar Teaching Institute of Public Health Croatia State General Laboratory Cyprus Czech Agriculture and Food Inspection Authority (CAFIA) Czech Republic UKZUZ (Central Institute for Supervising and Testing in Agriculture) Czech Republic Danish Veterinary and Food Administration Denmark DTU Food Denmark Agricultural Research Centre Estonia Finnish Food Safety Authority Evira Finland Laboratoire SCL de Rennes France Laboratoire des Pyrenees et des Landes France CVUA Rheinland Germany Federal Institute for Risk Assessment Germany Lower Saxony State Office for Consumer Protection and Food Safety Germany CVUA Sigmaringen Germany Feedstuffs Control Laboratory of Athens, Ministry of Rural Development & Food Greece General Chemical State Laboratory Greece Chemical State Laboratory, Division of Piraeus and the Aegean Greece
National Food Chain Safety Office, Food And Feed Safety Directorate, Food Toxicological NRL Hungary National Food Chain Safety Office, Food and Feed Safety Directorate, Feed Investigation National Reference Laboratory
Hungary
Public Analyst's Laboratory Dublin Ireland Ireland State Laboratory, Contaminants Department Ireland Azienda USL Toscana centro Italy Istituto Zooprofilattico Sperimentale del Mezzogiorno Italy IZSLER Italy ARPAL Italy ATS Val Padana Italy IZS Sicilia Italy Istituto Superiore di Sanità – National Reference Laboratory for Mycotoxins Italy Institute of Food Safety, Animal Health and Environment "BIOR" Latvia National Food And Veterinary Risk Assessment Institute Lithuania Laboratoire national de santé Luxembourg Public Health Laboratory Malta RIKILT - Wageningen University and Research Netherlands National Veterinary Research Institute Poland ASAE Portugal Institute for Hygiene and Veterinary Public Health Romania Regional Public Health Authority in Poprad Slovakia State veterinary and food institute Dolný Kubín, Veterinary and food institute in Košice" Slovakia National laboratory for health, environment and food Slovenia National Centre for Food (Spanish Consuming Affairs, Food Safety and Nutrition Agency Spain Consejeria de Desarrollo Rural y Rec. Naturales – Laboratorio de Sanidad Animal Spain GV. Conselleria de Sanidad Universal y Salud Pública. Centro de Salud Pública Spain CNTA Spain Laboratorio de Salud Pública de Albacete. Junta de Comunidades de Castilla-La Mancha Spain National Food Agency Sweden
3
National Veterinary Institute (SVA) Sweden Fera Science Ltd UK West Yorkshire Analytical Services UK Public Analyst Scientific Services Limited UK
4
List of abbreviations and definitions
AFB1 Aflatoxin B1 BEA Beauvericin CEN European Committee for Standardization DON Deoxynivalenol ELISA Enzyme-linked immunosorbent assay EN Enniatin EMD-IDMS Exact-Matching Double Isotope Dilution Mass Spectrometry EURL European Union Reference Laboratory FAO Food and Agriculture Organization FB1&2 Fumonisins B1 and B2 HPLC-FLD High performance liquid chromatography-fluorescence detection HPLC-UV(DAD) High performance liquid chromatography-ultraviolet/diode array detection IAC Immunoaffinity extraction column/clean-up ISO International Organization for Standardization JRC Joint Research Centre LC-HiResMS Liquid chromatography-high resolution mass spectrometry LC-MS/MS Liquid chromatography-tandem mass spectrometry GC-MS(/MS) Gas chromatography-mass spectrometry or tandem mass spectrometry LOD Limit of Detection LOQ Limit of Quantification MS Member States NRL National Reference Laboratory OCL Official Control Laboratory PT Proficiency Test QuEChERS Quick, Easy, Cheap, Effective, Rugged and Safe ZON Zearalenone
5
1 Introduction
Mycotoxins are products of fungal secondary metabolism produced by filamentous fungi that can infect agricultural commodities both in the field and during storage [1]. Over 400 mycotoxins are known nowadays, but just about 30 occur in food and feed [2]. Aflatoxins, trichothecenes, fumonisins, ochratoxin A, zearalenone, patulin and Alternaria toxins are considered to be of the greatest importance to human and animal health, and to have the biggest detrimental economic impact in food trade [2-4].
They can display a range of severe toxic effects in humans and animals. Aflatoxin B1 (AFB1) is the most potent natural carcinogen in experimental animals (rats), ochratoxin A (OTA) is nephrotoxic, fumonisins B1 and B2 (FB1 and FB2) exhibit neuro- or hepatotoxicity and carcinogenicity depending on the target species affected, deoxynivalenol (DON) shows immunotoxic effects, zearalenone (ZON) is an endocrine disruptor, binding to the oestrogen receptors; and T-2 and HT-2 toxins inhibit protein synthesis and are highly haematotoxic [1,2,5,6].
Nowadays, their co-occurrence and combined toxicity is gaining increased interest. The same fungus might produce different mycotoxins, and various fungi can affect the same crop. Exposure to several classes of mycotoxins often results in an additive effect, not excluding a possible synergistic interaction [6-8]. Maize is an example where several mycotoxins have been reported to occur simultaneously.
The Food and Agriculture Organization (FAO) has estimated that 25% of the world’s food crops are contaminated with mycotoxins, but the actual figures might well be much higher [9]. Kovalsky et al. [6] reported contamination rates varying between 7 and 79 % for B trichothecenes and 88 % for ZON, while enniatins (ENs) were ubiquitous.
Presently, AFs are regulated with maximum levels in 18 food categories, OTA in 13, DON in 9, ZON in 10 and fumonisins in 6 [10]. Indicative levels were established for the sum of T-2 and HT-2 toxins in unprocessed cereals and cereal products [11]. Food safety concerns have been extended recently to the so-called ‘‘emerging’’ mycotoxins such as ENs and beauvericin (BEA). ENs exhibit biological activity acting as enzyme inhibitors, are antifungal and antibacterial agents, and immunomodulatory substances [12]. BEA is cytotoxic and can induce apoptosis and DNA fragmentation. ENs and BEA act as ionophores, disturbing the pH and physiological ionic balance [13]. Enniatin B, the most prevalent EN, was found by Juan et al. in 70 % of the baby food samples at levels of up to 1100 µg kg-1 and in 44 % of the pasta samples at levels of up to 106 µg kg-1, while other authors reported contamination rates of between 50-90 % of the wheat, maize and barley samples with total concentrations of EN and BEA of up to 500 mg kg-1 [12].
The trends in food analysis go in the direction of developing multi-analyte methods, combining a generic sample preparation protocol with a highly selective instrumental analysis, such as liquid chromatography-mass spectrometry (LC-MS) [14,15]. This may allow monitoring for larger numbers of potential contaminants and revealing heretofore unknown potential hazards [3]. LC-MS-based multi-mycotoxin methods offer improved selectivity and sensitivity, a substantial reduction of the sample preparation, and simultaneous quantification and confirmation of the identity [1,2]. Despite the fact that the performance of the laboratories has enhanced over the recent years, improvements in accuracy, efficiency and the management of matrix effects are still needed [1,2]. A previous proficiency test (PT) highlighted that matrix-matched calibrations or calibrations using 13C-labelled mycotoxins as internal standards were essential for accurate mycotoxin quantification [1]. Nevertheless, neither all national reference laboratories (NRLs) use LC-MS methods for mycotoxin determinations nor they resort to the above tools for matrix compensation. Therefore, the continued evaluation of their proficiency is required. Besides, efforts should be undertaken to foster their analytical capability on the determination of emerging mycotoxins (e.g. ENs and BEA).
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2 Scope
As stated in Article 32 of Regulation (EC) No 882/2004 of the European Parliament and of the Council [16], one of the core duties of the EURL is to organise PTs for the benefit of the NRLs.
Given the fact that single mycotoxin PTs are only of relevance where a particular mycotoxin is regulated the conduction of multi-mycotoxin PTs is an elegant way of recognising the fact that for some food matrices a number of mycotoxins are potential contaminants. Therefore, a proficiency test including naturally contaminated test materials was organised covering all regulated mycotoxins in cereals, except ochratoxin A. Mycotoxins of emerging concern, such as ENs and BEA were also included to gauge the analytical capability and proficiency among the participants. The determination of enniatins A, A1, B, B1 and beauvericin was not mandatory but highly encouraged.
The proficiency test was addressed to all NRLs for mycotoxins and to appointed Official Control Laboratories (OCLs). Participation was mandatory and free of charge for the NRLs. Fifty-six laboratories from 28 Member States registered for the PT.
The EURL Mycotoxins performed the planning, execution and assessment of the measurement results based on the requirements laid down in ISO/IEC 17043:2010 [17]. Participants' results were evaluated using the ProLab software package (Quodata, Dresden, DE). The team that organised this PT is an ISO/IEC 17043:2010 accredited PT provider [18].
3 Confidentiality
The procedures used for the organisation of PTs are accredited according to ISO/IEC 17043:2010 [17] and guarantee that the identity of the participants and the information provided by them is treated as confidential. However, lab codes of the NRLs appointed in line with the Regulation (EC) No 882/2004 will be disclosed to DG SANTE upon request for (long-term) performance assessment.
4 Time frame
The PT was announced to the National Reference Laboratories by email and through the EURL Mycotoxins web page [18] on 15th July 2016. Registration for this PT was open until 02nd September 2016 (Annex 1). The participants were given six weeks after the dispatch of the samples (13th and 14th September 2016) for analysing them and reporting back the results together with the duly filled questionnaire. The deadline for reporting the results was 28th October 2016.
5 Materials
5.1 Preparation
The oat test material was produced by combining two contaminated oat batches with one blank oat. The corn material was produced by combining two low contaminated materials with a small amount of a highly contaminated corn supplied by Trilogy (Washington, USA). The materials were thoroughly homogenised, bottled and stored in the freezer until dispatch. Batches of approximately 5 kg of oat and 10 kg of corn were prepared and approximately 55 g and 100 g portions, respectively, were packed in amber plastic bottles. The materials were envisaged to contain as many regulated mycotoxins as possible, as well as enniatins and beauvericin. The contamination levels were in the range as commonly found in cereal samples.
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5.2 Homogeneity
For checking the homogeneity of the test materials, 10 units per material (oat and corn) were randomly selected from the production lot. Two independent determinations were performed per bottle using a liquid chromatography-isotope dilution tandem mass spectrometry (LC-ID-MS/MS) method that was collaboratively validated to be published as CEN standard. The determination of beauvericin was carried out separately, consisting of an extraction with a mixture of water:ethyl acetate 1:2 followed by salting-out with sodium sulfate and clean-up with a silica solid-phase extraction column. Both methodologies are described in the working instruction D-00797 of the JRC Geel. The order of measurements was randomised. Homogeneity was evaluated according to ISO 13528:2015 [19]. The materials proved to be adequately homogeneous (Annex 2).
5.3 Stability study
The stability study was conducted following an isochronous experimental design [20]: -70 °C was chosen as the reference temperature for sample storage. Stability was assessed at the following test temperatures: room temperature (≈20 °C), 4 °C and -18 °C. The time periods considered in this study were: 14, 28 and 55 days. The stability was evaluated according to the requirements of ISO 13528:2015 [19]. A linear regression was drawn for each tested temperature over the duration of the study, and the significance of the slope departure from zero at 95 % confidence level was verified (Annex 3). The materials proved to be adequately stable at room temperature, 4 °C and -18 °C for the period between dispatch (t=0) and the submission date of the last results (t=55 days). Based on a similar PT (mycotoxins in cereals) of 2013, the regulated mycotoxins should be stable in the present PT matrix during 1-2 days shipment without cooling.
5.4 Distribution
The test materials were dispatched in polystyrene boxes at ambient temperature on 13th and 14th September 2016. The samples were mostly received within 24 hours after dispatch. Storage was required to be at -18 °C until analysis. Each participant received: a) two test materials for analysis, packed in amber plastic bottles - Sample O-1## – oat (approx. 55 g) - Sample C-2## – corn (approx. 100 g) b) an accompanying letter with instructions on sample handling and reporting (Annex 4) d) a sample receipt form (Annex 5) and e) laboratory specific files for reporting with a lab code (by email).
6 Instructions to the participants
The scope of the PT and the instructions for sample handling and reporting of the results was communicated to the participants via an accompanying letter (Annex 4). The laboratories were required to report the mass fractions of the regulated mycotoxins and enniatins and beauvericin in µg kg-1 (mass as received) following their routine practices, accompanied by the measurement uncertainty (µg kg-1) for at least the regulated mycotoxins (k=2). Then, in the Questionnaire (Annex 6), participants were asked to mention whether the results were corrected for recoveries or not and to provide the recoveries figures (in %).
The results were reported by the participants using the RingDat software, which is part of the ProLab software [21]. Laboratory specific files generated by ProLab were sent to each laboratory by email. A detailed questionnaire was also included. The questionnaire was
8
intended to provide additional information on method-related aspects and laboratory capabilities to allow insights on potential individual and general results' trends as well as to improve the planning of future PTs.
Method-related details such as the type of extraction and clean-up protocols, chromatographic and detection conditions, calibration strategy and quality control; and performance parameters such as LODs and LOQs were requested.
Participants were informed about the shipment of the materials at ambient temperature and that upon arrival they should be transferred to -18 °C. Participants were also encouraged to perform the analysis as soon as possible to allow enough time for data treatment, get acquainted with the software for reporting and resolve any unexpected instrumental issue.
7 Reference values and their uncertainties
The assigned values of the regulated mycotoxins in the test materials and their uncertainties were established by Exact-Matching Double Isotope Dilution Mass Spectrometry (EMD-IDMS) at JRC-Geel (Table 2). This methodology is considered to provide the highest degree of accuracy of the assigned values [22].
The reference values for the enniatins were obtained by standard addition, due to the lack of the corresponding isotope-labelled standards required for performing EMD-IDMS. These values should be regarded as indicative.
Table 2. Assigned values of the analytes and their associated expanded uncertainties in oat and corn test items
Matrix Analyte Technique Assigned value (µg kg-1)
U (k=2) (µg kg-1)
Corn
Deoxynivalenol EMD-IDMS 611 32
Aflatoxin B1 EMD-IDMS 10.61 0.65
Zearalenone EMD-IDMS 161.6 8.8
Fumonisin B1 EMD-IDMS 768 50
Fumonisin B2 EMD-IDMS 224 16
Oat
T-2 toxin EMD-IDMS 70.3 2.1
HT-2 toxin EMD-IDMS 150.3 9.5
Enniatin B Stand. Add. 36.4 2.8 1
Enniatin B1 Stand. Add. 26.3 2.2 1
Enniatin A1 Stand. Add. 7.95 0.84 1
Enniatin A Stand. Add. <LOQ −
U - expanded uncertainty of the assigned value 1 A conservative approach was adopted in the absence of information about the uncertainty of the purity of the calibrants
8 Evaluation of the results
8.1 General observations
Fifty-six participants from 28 countries registered for the exercise and 53 datasets were reported back. Thirty-six laboratories were NRLs for mycotoxins and 17 were OCLs. Both NRLs for food and feed from Czech Republic, Denmark, Greece, Ireland, Hungary, Slovakia and Sweden have participated in this PT.
It was intended that the test materials distributed would contain the widest possible range of regulated mycotoxins and additionally, enniatins and beauvericin. The
9
concentrations of ochratoxin A and enniatin A were, however, too low to be reliably quantified and most probably fall below the LOQs of routine analytical methods. The mycotoxins that were requested to be analysed were split between the two materials to limit the analytical work of the laboratories, although such materials may contain other mycotoxins.
The laboratories were free to use their method of choice reflecting their routine procedures. More than half of the laboratories (33) used LC-MS/MS-based multi-mycotoxin methods while many laboratories still used HPLC-UV(DAD) for the determination of DON, and HPLC-FLD was the preferred technique for the determination of AFB1.
8.2 Scores and evaluation criteria
The individual laboratory performance was assessed in terms of z- and zeta- (ζ) scores following ISO 13528:2015 [19]. The following formulas were used:
� = ��������� Equation 1
= ������������� ���� Equation 2
where:
���� is the measurement result reported by a participant
���� is the reference value (assigned value)
���� is the standard uncertainty reported by a participant
���� is the standard uncertainty of the reference value
�� is the standard deviation for proficiency assessment (target standard deviation)
�� was calculated as 22 % of the assigned value. The coefficient derived from the Horwitz equation for a mass fraction of 120 µg kg-1 (�� = 0.22!) was applied regardless of the magnitude of the mass fraction of each given analyte. Data collected in previous PTs indicated that this coefficient often closely resembles the reproducibility standard deviation of the participants' data.
The z-score compares the participants' deviation from the reference value with the target
standard deviation accepted for the proficiency test, ��. The z-score is interpreted as:
|z| ≤ 2 indicates satisfactory performance
2 < |z| < 3 indicates questionable performance
|z| ≥ 3 indicates unsatisfactory performance
The zeta (ζ)-score indicates whether the participants' estimate of the uncertainty is consistent with the observed deviation from the assigned value. The ζ-score is the most relevant evaluation parameter, as it includes all parts of a measurement result, namely the expected value, its uncertainty as well as the uncertainty of the assigned value.
10
The interpretation of the ζ-score is similar to the interpretation of the z-score:
|ζ| ≤ 2 indicates satisfactory performance
2 < |ζ| < 3 indicates questionable performance
|ζ|≥3 indicates unsatisfactory performance
An unsatisfactory performance based on a |ζ|-score ≥3 might be due to an underestimation of the uncertainty, a large deviation from the reference value or to a combination of the two factors.
8.3 Laboratory results and scoring
The statistical evaluation of the results was performed using the ProLab software [21]. Z-and ζ-scoring was based on the reference values (and respective uncertainties) assigned by EMD-IDMS instead of the consensus values (robust mean). The robust mean and the reproducibility standard deviation were computed according to the Algorithm A of ISO 13528:2015, and are given in Table 3 just for information purposes [19].
No performance scoring was attempted for the enniatins and beauvericin, as the reduced number of participants (max. 15) limited the robustness of the consensus estimation of the actual mass fraction while the calibration standards used in the organiser's estimations were not accompanied by the uncertainty of the purity. The two estimations above for enniatin B and A1 deviated 6.6 and 7.6 % from each other, respectively, which is in the range of their uncertainties, while the estimations for enniatin B1 deviated by 43 %. The mass fraction of enniatin A is below the method's LOQ, while, in general, the calibrations for beauvericin didn't meet the requirement of r2>0.99 due to the dispersion of the results. The values supplied should be seen as indicative, and any technical judgement on the analytical performance is reserved to each of the participants.
83.7 % of the results reported by the participants were rated with satisfactory z-scores (|z|≤ 2), taking into consideration all regulated mycotoxins requested in the two matrices (DON, AFB1, ZON, FB1, FB2, HT-2 and T-2) (see figure 1).
6.1 % of the results fell into the unsatisfactory range with |z|≥ 3
Figure 2 shows that the performance of the laboratories analysing DON, AFB1, ZON, FB1 and FB2 present in the corn material was better than analysing HT-2 and T-2 (for which just a Commission Recommendation exists) in oat. Additionally, the number of laboratories that reported results for HT-2 (39) and T-2 (40) was lower than for AFB1 (52), ZON (49) and DON (48), which are strictly mandatory determinations in a variety of food matrices.
The determination of HT-2 and T-2 toxins was almost exclusively done by LC-MS/MS, which requires an appropriate compensation of the matrix effects by using the corresponding isotopologues, opposite to other mycotoxins which can be analysed by robust techniques such as HPLC-FLD after immunoaffinity clean-up (IAC).
Figures 3 and 4 present an overview of the individual z-scores assigned to the results provided by each laboratory. The longer the triangles, the larger were the differences to the assigned values. Blue triangles represent z-scores in the satisfactory range, yellow triangles in the questionable range and red triangles in the unsatisfactory performance range. The unsatisfactory scores are shown next to the red triangles.
The numerical values of the calculated z-scores and ζ-scores are compiled in Tables 4 and 5. All z- and ζ-scores in the satisfactory performance range are shown with a green background; those in the questionable range are displayed with a yellow background and scores indicating unsatisfactory performance are presented with a light-red background.
11
Figure 1. Overall distribution of the z-scores obtained by the participants for the regulated mycotoxins in the corn and oat materials
Figure 2. Distribution of the z-scores for the regulated mycotoxins present in the corn (left) and oat (right) materials
Distribution of Z-Scores
Z-Scores543210-1-2-3-4
Ab
solu
te fr
equ
en
cy
40
38
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
Ring test: PT 2016 MULTITOXIN7 Measurands53 Laboratories2 Samples295 Z-Scores
The graphical representations of the sigmoidal distribution of the results (µg kg-1) for each combination of analyte/sample are given in Figure 5. Reported values are shown as bars. The green line corresponds to Xref; the green shadow covers the boundary of the reference interval (Xref ± uref), and the red lines mark the boundary of the target interval (Xref ± 2σ). Green bars represent results with |z-score| ≤2, yellow bars represent results with 2<|z-score|<3, while the red bars represent results with |z-score|≥3.
Figure 3. Individual laboratory z-scores for the regulated mycotoxins in corn.
Figure 4. Individual laboratory z-scores for the regulated mycotoxins in oat.
A summary of the statistical evaluation of the results of the regulated mycotoxins, enniatins and beauvericin is presented in Table 3. The robust standard deviations of the reported results for AFB1, DON and ZON were close or below the target standard deviation (22 %) while they were somewhat higher for FB1 and FB2. On the other hand, the robust standard deviations for HT-2 and T-2 toxins are clearly above the target standard deviation (45 and 33 %, respectively). This finding reflects the maturity of the laboratories analysing these mycotoxins. Not all NRLs submitted results for the HT-2 and T-2 toxins and efforts are still needed to improve the analytical performance.
The limited number of laboratories reporting results for enniatins and the fact that these mycotoxins are not part of a routine monitoring may explain the higher robust standard deviation that was calculated (from 41 to 60 %). At the highest extreme is beauvericin, which suffers from considerable signal variability from run to run when analysed by LC-MS/MS. Presently, there are no commercially available isotope-labelled internal standards that can effectively compensate these analytical inconsistencies.
Annex 7 shows the individual kernel density plots for the mycotoxins covered by the PT. The confidence intervals of the robust means calculated from the participants' results overlap with the confidence intervals of the assigned values for all the analytes, except for the T-2 toxin.
Z-Score-3 0 3 -3 0 3
Measurand
T-2 HT-2
Labo
rato
ry
LC 0001LC 0005
LC 0007LC 0014
LC 0015LC 0018
LC 0020LC 0021LC 0022
LC 0024LC 0026
LC 0027LC 0028LC 0030
LC 0031LC 0032
LC 0033LC 0034
LC 0035
LC 0036LC 0037
LC 0038LC 0039
LC 0040LC 0041
LC 0043LC 0045
LC 0046
LC 0047LC 0048
LC 0049LC 0050
LC 0051LC 0052
LC 0054LC 0055LC 0056
5.2
6.6
5.6
5.6
-4.1
-3.1
-3.0
-3.7
-4.4
14
Table 3. Summary statistics of the results submitted for the regulated mycotoxins, enniatins and beauvericin in corn and oat
CORN OAT
Units DON AFB1 ZON FB1 FB2 HT-2 T-2 ENB ENB1 ENA1 ENA BEA
1 No reliable value could be established, see chapter 8.3 on page 10 for details. 2 A conservative approach was adopted in the absence of information about the uncertainty of the purity of the calibrants
15
Table 4. Reported results and respective z-scores and ζ-scores in the corn test material
Sample: CEREALS OATMeasurand ENNIATIN BMethod: ISO 5725-5 (Alg. A+S)Number of laboratories in calculation: 13
Mean value: 34.1 µg/kgRel. reproducibility s.d.: 41.72%
Mea
n
<10.
0 (Q
L)
<15.
0 (Q
L)
<50.
0 (Q
L)
Laboratory
LC00
56
LC00
43
LC00
17
LC00
47
LC00
05
LC00
31
LC00
48
LC00
40
LC00
15
LC00
34
LC00
01
LC00
18
LC00
14
LC00
39
µg/
kg
50
40
30
20
10
0
Sample: CEREALS OATMeasurand ENNIATIN B1Method: ISO 5725-5 (Alg. A+S)Number of laboratories in calculation: 11
Mean value: 17.0 µg/kgRel. reproducibility s.d.: 40.85%
Mea
n
<10.
0 (Q
L)
<15.
0 (Q
L)
<50.
0 (Q
L)
Laboratory
LC00
56
LC00
47
LC00
15
LC00
31
LC00
43
LC00
18
LC00
40
LC00
17
LC00
01
LC00
05
LC00
14
LC00
39
LC00
34
µg/
kg
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
Sample: CEREALS OATMeasurand ENNIATIN A1Method: ISO 5725-5 (Alg. A+S)Number of laboratories in calculation: 8
Mean value: 7.4 µg/kgRel. reproducibility s.d.: 60.01%
Mean
<2.0
(Q
L)
<10.
0 (Q
L) <15.
0 (Q
L)
Laboratory
LC00
43
LC00
26
LC00
31
LC00
47
LC00
40
LC00
56
LC00
18
LC00
55
LC00
15
LC00
05
LC00
34
LC00
39
LC00
01
LC00
14
LC00
48
LC00
17
µg/
kg
35
30
25
20
15
10
5
0
Sample: CEREALS OATMeasurand BEAUVERICINMethod: ISO 5725-5 (Alg. A+S)Number of laboratories in calculation: 14
Mean value: 15.8 µg/kgRel. reproducibility s.d.: 87.68%
Mea
n
<15.
0 (Q
L) <25.
0 (Q
L)
21
The plausibility of the uncertainty statements of the laboratories was assessed by classifying every reported uncertainty into one of the three groups (see column C in Tables 4 and 5) according to the following rules.
The standard measurement uncertainty of a result (u(xi)) is most likely to fall within a range between a minimum and a maximum uncertainty (case "a": umin ≤ u(xi) ≤ umax). The minimum uncertainty (umin) is set for the respective analyte to the standard uncertainty of the assigned value (u(xpt)). This is based on the assumption that it is unlikely that a laboratory carrying out the analysis on a routine basis would determine the measurand with a smaller measurement uncertainty than that achieved in the experiments for the characterisation of the test material, which was based on EMD-IDMS. The maximum uncertainty is set to the standard deviation accepted for the assessment of results (σpt). Consequently, case "a" becomes: u(xpt) ≤ u(xi) ≤ σpt.
If u(xi) is smaller than u(xpt) (case "b": u(xi) < u(xpt)) the laboratory might have underestimated its measurement uncertainty.
If u(xi) is larger than σpt (case "c": u(xi) > σpt), the laboratory might have overestimated its measurement uncertainty or applied an analytical method that was not fit-for-purpose. Both cases require amendment.
The rate of the satisfactory ζ-scores is lower than the one for z-scores. The participants in categories "b" and "c" are encouraged to assess their uncertainty estimation in line with the above observations. The uncertainty is an integral part of the measurement result and has major implications on the assessment of the compliance of food according to the European Union legislation. Annex 8 presents the sigmoidal distribution of the results associated with the respective uncertainties (k=2).
9 Evaluation of the questionnaire
The questionnaire distributed to the participants has provided very useful information concerning the approaches and capabilities of the laboratories in the determination of regulated mycotoxins, enniatins and beauvericin in cereals (Annex 6).
The questionnaire will be discussed in two parts:
1) the first part will address the answers regarding the previous experience of the participants and general organisational matters: questions Q.1-3 and Q.19-29.
2) the second part will deal with the outcome of the answers concerning analytical features (questions Q.4-16 and Q.18) and will present the validation data of the methods used by the PT participants.
9.1 Experience and organisational aspects
The participants were asked to classify their yearly work load on the analysis of mycotoxins in 3 categories (Q.1). The results are summarised in Figure 6. The most frequently analysed mycotoxins were: aflatoxins, deoxynivalenol and zearalenone, while the least were the enniatins and beauvericin, both in terms of the number of samples (mainly <50) and the number of laboratories that conducted it (18). The type of matrices was diverse: figs, nuts, spices, cereals/flour and cereal products, feed products, peanuts, baby-food, etc. (Q.2) (Annex 9). Of the 46 laboratories that answered the question on accreditation (Q. 3), 87 % hold an accreditation for measuring aflatoxin B1, 83 % for deoxynivalenol and 80 % for zearalenone. 35 % of the accredited methods were multi-mycotoxin procedures (Table 6).
Presently, 79 % of the participants don't analyse enniatins and beauvericin, and out of these, 35 % don't plan to implement this determination in the near future (Q. 19 and 20). Still, another 38 % foresee implementing a suitable analytical procedure in the mid-term future (within 1-2 years).
22
Figure 6. Number of mycotoxin determinations performed by the laboratories on a yearly basis.
Table 6. Number of laboratories accredited for the determination of mycotoxins in food.
AfB1 DON ZON FB1 FB2 HT-2 T-2 ENs BEA Multitoxin
method
N. labs 40 38 37 23 23 30 29 4 3 16
% 87 83 80 50 50 65 63 9 7 35
The majority of the participants did not experience any difficulties during this PT (Q.21, Table 7). Those who mentioned issues related them mainly to the sensitivity of their analytical instruments and the complexity of the matrices (Q.22, Table 8). For 92 % of the participants the time allowed for reporting the results was adequate (Q.24), while 84 % also found the amount of sample provided as sufficient for the analysis (Q.25). The vast majority of the participants didn't face any difficulties with the software for reporting the results (Q. 27), and they were all happy with the instructions to carry-out the PT (Q.28) (Annex 4). About 65 % of the participants completed the PT analyses in one week or less (Q.26). Despite the general satisfaction with the layout of the PT and the information provided, some participants took the opportunity to raise some remarks in the Comments section (Q.29). A compilation can be found in Table 9.
The most effective route to spread information on upcoming PTs still seems to be by direct contact, via email or during the annual workshops (Q.23, Table 10).
Table 7. Answers related to the experience of the participants during this PT and the evaluation of organisational aspects.
Response
Q.21 Q.24 Q.25 Q.27 Q.28
NO Nr. 40 4 8 43 0
% 77 8 16 91 0
YES Nr. 12 48 43 4 48
% 23 92 84 9 100
12
%
22
%66
%
Aflatoxins: <50
Aflatoxins: 50-100
Aflatoxins: >100
31
%
26
%
43
%
Deoxynivalenol: <50
Deoxynivalenol: 50-100
Deoxynivalenol: >100
33
%
30
%
37
%
Zearalenone: <50
Zearalenone: 50-100
Zearalenone: >100
49
%27
%
24
%
Fumonisins: <50
Fumonisins: 50-100
Fumonisins: >100
34
%
29
%
37
%
T-2 and HT-2: <50
T-2 and HT-2: 50-100
T-2 and HT-2: >100
78
%
22
%
ENs and bea: <50
ENs and bea: 50-100
ENs and bea: >100
23
Table 8. Analytical difficulties experienced running the PT Sensitivity loss of 80 % for the multimethod Recovery rates for enniatins and BEA were very low. We never had rates lower than about 90 % for the last 3 years in equal matrices! Probably due to the samples provided? No, for the routine samples, but yes for new ones - sensitivity of the instrument, insufficient clean-up
not the right matrix for calibration We have no LC MSMS, and for example we had problems with derivatisation of the T2/HT2 toxins (both the standard solutions and the samples) complex matrix, therefore insufficient clean-up Probably depending to the thinness of the sample particle it was impossible to obtain a clear test solution especially for DON-ZEA-T-2-HT-2 analysis. Carry-over between subsequent LC-MS/MS runs was observed for fumonisin B1&B2 and beauvericin, which required thorough rinsing Sensitivity of the instrument, pump leak, difficulties with recovery estimation ...
matrix effects for oat sample sensitivity problems on our MS/MS for enniatins and beauvericin
Some matrix effects due to insufficient clean-up of the PT samples
problem of filtration for Afla B1 in corn sample only
Sensitivity of the instrument
Table 9. Comments submitted by the participants Besides Aflatoxin B1 in corn, there was Aflatoxin B2. The sum of aflatoxins is 9.83 µg/Kg.
The results for all enniatins and beauvericin are <LOD, where LOD was estimated as 5 ug/kg
Enniatin-amount is quite low in comparison to our routine samples. For Fumonisins, the result is the sum of Fum B1+B2 together, but there is no possibility to type together, so I put it in the column for Fum B1. For T2 and HT2, I didn't obtain adequate recoveries, so I am not sending the results. Time and sample amount were enough for routine analyses, for new - I would appreciate more time and more samples to work on methods. We tried to analyse enniatins and beauvericin too, but the technique was not sensitive enough. These samples were analysed with the newly validated multitoxin method. Will be accredited in few weeks. The high uncertainty values obtained in some cases (AFB1 and FB1) do not exclude a lack of sufficient homogeneity in the sample. Thank you for the opportunity to participate No data reported for beauvericin, enniatins and fumonisins. This is due to the absence of recovery data. The chosen spiking level was not sufficient to significantly add to the residue already present, and therefore recovery values could not be calculated. From the measurands requested, the only 3 mycotoxins which are reported are those which are analysed in the laboratory; AFLA, DON and ZON. Our laboratory does not have an LC-MS/MS and future analysis of the mycotoxins not reported here will depend on whether such instrumentation is procured. We were not able to provide results for enniatins and beauvericin since with our current MS/MS detector (Waters TQD), it was not possible to implement a multimycotoxin method up to now. We hope to do this after installation of our new Xevo-TQ-S next month. We don't use the terminology of "LOD/LOQ", for our routine analyses we report the "reporting limit" to the customer. This (usually) coincides with the LOQ. Hence we didn't report an LOD. Unfortunately, we have no experience for enniatin and beauvericin.
The method used is screening The recoveries indicated are "apparent recoveries", i.e to account for both extraction recovery and matrix effects
Beauvericin and Enniatins integrated relative to the internal standard associated with T-2.
Table 10. Information source about the PT on multi-mycotoxins
Invitation/announcement of the PT %
Invitation by email 73 Through the EURL Mycotoxins website 0 During the EURL workshop for the NRLs on mycotoxins 13 By the NRL in your country 23
24
9.2 Overview of the analytical methodologies
A considerable number of laboratories resorted to LC-MS/MS-based multi-mycotoxin methods for analysing the distributed PT samples (see Table 11). This was the only methodology that allowed analysing all the requested analytes, including regulated and emerging mycotoxins (enniatins and beauvericin). LC-MS/MS analysis was mostly preceded by a sample preparation of the "dilute and shoot" type (14 cases), but QuEChERS, IACs and SPE also found application in 4 to 5 cases each. Pressurised liquid extraction (PLE) was employed just by a single laboratory. HPLC-UV(DAD) was used by 11 participants to analyse DON while HPLC-FLD was used chiefly to analyse AFB1 but also ZON, fumonisins, HT-2 and T-2. These two techniques were almost invariably preceded by an IAC clean-up. LC coupled with high-resolution MS (HiResMS) was used as a screening method. One laboratory reported the sum of FB1 and FB2 as their ELISA did not allow individuating the analytes. Another laboratory also using ELISA reported results <LOQ for FB1, FB2, HT-2 and T-2.
Of the laboratories that employed LC-MS/MS methods, 62 % used 13C-labelled internal standards (Q.10) which were added in the majority of cases (88 %) after the extraction (Q.11). The analysis of calibration standards prepared in the pure solvent was the dominant strategy (78 %, Q.12) in good correlation with the use of either individual mycotoxin methods or multi-methods employing 13C internal standards.
Most of the laboratories (88 %) have estimated the methods´recoveries based on spiking experiments while the remaining used certified reference materials or an alternative strategy (Q.14). Nevertheless, in only 71 % of the cases, the results were corrected for recoveries (Q.15). The use of certified reference materials for quality control is not a common practice for the laboratories, but about 30 % of them mentioned the use of reference/quality control materials from FAPAS or Trilogy (Q.16).
The preferred approach for estimating the measurement uncertainty was using “initial method validation data” accounting for 53 % of the participants (Q.13).
Annex 9 presents a compilation of the main analytical conditions of the methods used by the participants such as the type of method, extraction conditions, clean-up, LC-MS acquisition settings, quantification strategy, amongst others. Annex 10 compiles some important analytical figures of merit (recoveries, LODs and LOQs). The median recoveries for all analytes varied from 91 to 98 %. In general, the sensitivity of the methods was sufficient to analyse the mycotoxin levels present in the PT materials.
Table 11. Analytical methods and number of laboratories that have adopted them for participating in the PT
DON AFB1 ZON FB1 FB2 HT-2 T-2 EN A EN A1 EN B EN B1 BEA
LC-MS/MS 33 24 30 28 27 31 32 13 12 15 13 15
HPLC-UV(DAD) 11 - - - - - - - - - - -
HPLC-FLD - 26 15 9 9 3 3 - - - - -
ELISA 2 1 3 3 3 3 3 - - - - -
LC-HiRes 1 1 1 1 1 1 1 - - - - -
GC-MS(/MS) 1 - - - - 1 1 - - - - -
10 Conclusions
A PT was organised by the EURL mycotoxins covering 12 mycotoxins spread over two test materials (oat and maize) to allow the participants to test their multi-mycotoxin procedures. Laboratories were also encouraged to report results for enniatins and beauvericin which, although not mandatory, was an important focus of this PT and of special interest for DG SANTE.
A total of 53 laboratories submitted results providing from 39 to 52 datasets for the
25
regulated mycotoxins and a maximum of 15 datasets for the enniatins and beauvericin. Overall, 83.7 % of the results reported by the participants for the regulated mycotoxins were classified as satisfactory. The rate of satisfactory z-scores for the individual mycotoxins was ranked as follows: AFB1 - 94 %, DON – 91 %, ZON - 89 %, FB1 – 87 %, FB2 – 78 %, T-2 – 75 % and HT-2 – 64 %. Twelve laboratories had satisfactory performance in all the 7 regulated mycotoxins while 8 additional laboratories had one questionable result.
Eleven laboratories reported results for all enniatins and beauvericin. The consensus values for enniatin B and A1 were very close to the EURL’s estimate although no z-scoring was attempted as the reference values didn't meet the required level of accuracy to make sound statements.
Up to 33 laboratories used LC-MS/MS for analysing a combination of mycotoxins while 11 laboratories could analyse all the requested mycotoxins (12). HPLC-UV(DAD) was used by 11 laboratories to analyse DON exclusively. HPLC-FLD was the technique of choice for analysing AFB1 (26 laboratories) although it was also selected for analysing ZON (15 laboratories) and fumonisins (9 laboratories). ELISA was employed by 3 laboratories while GC-MS/MS and LC-HiResMS were used by a single laboratory each. The determination of mycotoxins by LC-MS/MS often included a straightforward sample preparation based on "extract, dilute & shoot", whereas HPLC-UV(DAD), HPLC-FLD and LC-HiResMS were preceded by an IAC clean-up.
No bias was observed when comparing LC-MS/MS-based multi-mycotoxin procedures and analyte-specific protocols based on the t-student test. The reproducibility standard deviation of the multi-mycotoxin and analyte-specific procedures was equivalent.
The aim of this PT on providing insight on the performance of multi-mycotoxin methods was successfully achieved as 24 laboratories determined the whole range of regulated mycotoxins. Some laboratories could also analyse enniatins and beauvericin although technical difficulties such as inappropriate recoveries and sensitivity issues hampered others to contribute as well.
26
References
[1] A.D. Girolamo, B. Ciasca, J. Stroka, S. Bratinova, M. Pascale, A. Visconti, V.M.T. Lattanzio, Performance evaluation of LCeMS/MS methods for multi-mycotoxin determination in maize and wheat by means of international Proficiency Testing, Trends in Analytical Chemistry, 86 (2017) 222-234.
[2] K. Zhang, J.W. Wong, A.J. Krynitsky, M.W. Trucksess, Perspective on advancing FDA regulatory monitoring for mycotoxins in foods using liquid chromatography and mass spectrometry., Journal of AOAC International, 99 (2016) 890-894.
[3] J. Stroka, C.M. Maragos, Challenges in the analysis of multiple mycotoxins, World Mycotoxin Journal, 9 (2016) 847-861.
[4] S. Monbaliu, C.V. Poucke, C.V. Peteghem, K.V. Poucke, K. Heungens, S.D. Saeger, Development of a multi-mycotoxin liquid chromatography/tandem mass spectrometry method for sweet pepper analysis., Rapid Communications in Mass Spectrometry, 23 (2009) 3-11.
[5] A.T. Åberg, A. Solyakov, U. Bondesson, Development and in-house validation of an LC-MS/MS method for the quantification of the mycotoxins deoxynivalenol, zearalenone, T-2 and HT-2 toxin, ochratoxin A and fumonisin B1 and B2 in vegetable animal feed., Food Additives and Contaminants: Part A, 30 (2013) 541–549.
[6] P. Kovalsky, G. Kos, K. Nährer, C. Schwab, T. Jenkins, G. Schatzmayr, M. Sulyok, R. Krska, Co-occurrence of regulated, masked and emerging mycotoxins and secondary metabolites in finished feed and maize - an extensive survey, Toxins, 8 (2016) 363.
[7] S. Monbaliu, C.V. Poucke, C. Detavernier, F. Dumoulin, M.V.d. Velde, E. Schoeters, S.V. Dyck, O. Averkieva, C.V. Peteghem, S.D. Saeger, Occurrence of mycotoxins in feed as analyzed by a multi-Mycotoxin LC-MS/MS method, Journal of Agricultural & Food Chemistry, 58 (2010) 66-71
[8] L. Anfossi, C. Giovannoli, C. Baggiani, Mycotoxin detection, Current Opinion in Biotechnology, 37 (2016) 120-126.
[9] V. Aiko, A. Mehta, Occurrence, detection and detoxification of mycotoxins, Journal of Biosciences, 40 (2015) 943-954.
[10] EC, Commission Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs, of 19 December 2006, and successive amendments, Official Journal of the European Union, L 364/5 (2006).
[11] EC, Commission Recommendation on the presence of T-2 and HT-2 toxin in cereals and cereal products, of 27 March 2013, Official Journal of the European Union, L 91/12 (2013).
[12] C. Juan, J. Mañes, A. Raiola, A. Ritieni, Evaluation of beauvericin and enniatins in Italian cereal products and multicereal food by liquid chromatography coupled to triple quadrupole mass spectrometry, Food Chemistry, 140 (2013) 755-762.
[13] M. Bolechov, K. Benesová, S. Belaková, J. Caslavsky, M. Pospíchalová, R. Mikulíková, Determination of seventeen mycotoxins in barley and malt in the Czech Republic, Food Control, 47 (2015) 108-113.
[14] H.G.J. Mol, P. Plaza-Bolan, P. Zomer, T.C.d. Rijk, A.A.M. Stolker, P.P.J. Mulder, Toward a generic extraction method for simultaneous determination of pesticides, mycotoxins, plant toxins, and veterinary drugs in feed and food matrixes, Analytical Chemistry, 80 (2008) 9450–9459.
[15] M. Sulyok, R. Krska, R. Schuhmacher, Application of an LC–MS/MS based multi-mycotoxin method for the semi-quantitative determination of mycotoxins occurring in different types of food infected by moulds, Food Chemistry, 119 (2010) 408–416.
27
[16] EC, Regulation (EC) No 882/2004 of the European Parliament and of the Council on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules, Official Journal of the European Union, L 165 (2004) 1-141.
[17] ISO/IEC 17043:2010 - Conformity assessment - General requirements for proficiency testing.
[18] JRC Geel. EURL for mycotoxins. Inter-laboratory comparisons. Available from: https://ec.europa.eu/jrc/en/eurl/mycotoxins/interlaboratory-comparisons.
[19] ISO 13528:2015; Statistical methods for use in proficiency testing by interlaboratory comparisons.
[20] A. Lamberty, H. Schimmel, J. Pauwels, The study of the stability of reference materials by isochronous measurements, Fresenius J Anal Chem, 360 (1998) 359–361.
[21] PROLab Plus - Software for PT programs and collaborative studies, Quodata, Dresden, Germany; http://quodata.de/en/software/for-interlaboratory-tests.html.
[22] L.G. Mackay, C.P. Taylor, R.B. Myors, R. Hearn, B. King, High accuracy analysis by isotope dilution mass spectrometry using an iterative exact matching technique, Accreditation and Quality Assurance, 8 (2003) 191-194.
28
List of Figures
Figure 1. Overall distribution of the z-scores obtained by the participants for the regulated mycotoxins in the corn and oat materials ..................................................11
Figure 2. Distribution of the z-scores for the regulated mycotoxins present in the corn (left) and oat (right) materials ...............................................................................11
Figure 3. Individual laboratory z-scores for the regulated mycotoxins in corn. ............12
Figure 4. Individual laboratory z-scores for the regulated mycotoxins in oat. ..............13
Figure 5. Sigmoidal plots of individual laboratory results reported for the regulated mycotoxins, enniatins and beauvericin in corn and oat. .............................................18
Figure 6. Number of mycotoxin determinations performed by the laboratories on a yearly basis. ........................................................................................................22
Table 2. Assigned values of the analytes and their associated expanded uncertainties in oat and corn test items .......................................................................................... 8
Table 3. Summary statistics of the results submitted for the regulated mycotoxins, enniatins and beauvericin in corn and oat ................................................................14
Table 4. Reported results and respective z-scores and ζ-scores in the corn test material ..........................................................................................................................15
Table 5. Reported results and respective z-scores and ζ-scores in the oat test material 17
Table 6. Number of laboratories accredited for the determination of mycotoxins in food. ..........................................................................................................................22
Table 7. Answers related to the experience of the participants during this PT and the evaluation of organisational aspects. .......................................................................22
Table 8. Analytical difficulties experienced running the PT ........................................23
Table 9. Comments submitted by the participants ...................................................23
Table 10. Information source about the PT on multi-mycotoxins................................23
Table 11. Analytical methods and number of laboratories that have adopted them for participating in the PT ...........................................................................................24
Ring test : PT 2016 MULTITOXIN (29 questions, 1277 answers)
Nr. Cue Question Answers
1 Samples per year How many samples does your laboratory approximately analyse for the following mycotoxins per year? Aflatoxins, enniatins and beauvericin, T-2 and HT-2, deoxynivalenol, zearalenone, fumonisins
51 Answers
2 Matrices Which food or feed matrices does your laboratory analyse most frequently for mycotoxins on a routine basis?
50 Answers
3 Accreditation
Is your laboratory accredited for the determination of any of the following mycotoxins in cereals? Aflatoxin B1, fumonisin B1, fumonisin B2, deoxynivalenol, zearalenone, T-2, HT-2, enniatins, beauvericin, multitoxin method
46 Answers
4 Multitoxin / individual method Did you use a multitoxin method or individual methods? 53 Answers
5 Analytical method Please indicate the acronym of the analytical method used for each mycotoxin or group of mycotoxins analysed (e.g., DON - IAC-HPLC-DAD)
53 Answers
6 Reference official method Please indicate the reference of the official method (if applicable) used to analyse each of the mycotoxins 37 Answers
7 Extraction conditions Please describe the extraction conditions or give a bibliographic reference of the SOP, in case you have used a multimethod
43 Answers
8 Clean-up For each mycotoxin, please indicate the brand of the immunoaffinity column or SPE column used for sample clean-up (if applicable) 45 Answers
9 MS conditions In case you have applied a LC-MS/MS multimethod, please indicate the MRM transitions used for quantification (e.g., DON - ESI+ m/z 297>249) 36 Answers
10 Isotope labelled Int Standards
In case you have applied a LC-MS/MS multimethod, did you use isotope-labelled internal standards? Please indicate which? 36 Answers
11 Addition of ISTD If applicable, did you add the internal standards? 21 Answers
12 Calibration approach Which type of calibration approach did you follow? Standards in pure solvent / Matrix matched calibration. Distinguish by mycotoxin, if needed. 49 Answers
13 Approach method uncertainty How have you estimated the method uncertainty? 51 Answers
14 Recovery estimate How did you estimate the method's recovery? 49 Answers
15 Recovery correction The results submitted were? 47 Answers
16 Use of CRMs Do you use Certified Reference Materials for mycotoxin analysis? Please specify the mycotoxins, matrices and suppliers of the CRMs 46 Answers
17 Suppliers of standards Which were the suppliers of the mycotoxin standards used for this Proficiency test 47 Answers
18 Special precautions Do you take special precautions to avoid the loss of analytes (e.g., acid washing of the glassware, amber glassware and protection from daylight, etc.)? Please indicate for which mycotoxins
48 Answers
19 Analysis of enniatins and beau
Did you analyse before enniatins and beauvericin in cereal samples? In case YES, for how long?
41 Answers
20 Implementation Enniatins In case you you don’t analyse enniatins and beauvericin, do you plan to implement the method in the near future? 32 Answers
21 Difficulties Did you have major difficulties analysing the distributed samples? 52 Answers
22 Which difficulties If Yes, please specify which? e.g. sensitivity of the instrument; pumps pressure; chromatographic resolution; tedious sample preparation; complex matrix, insufficient clean-up, etc.
17 Answers
23 PT announcement How were you informed about this Proficiency Test? 52 Answers
24 Time for reporting Was the time allowed for reporting the results adequate? 52 Answers
25 Sufficient sample Was the sample amount dispatched sufficient for the analyses? 51 Answers
26 Time spent for the PT How much time did you spend overall to analyse the samples, treat data and report? 51 Answers
27 Problems with Prolab/RingDat
Did you have any problems using the ProLab/RingDat platform for results reporting? If Yes, describe which? 47 Answers
28 Instructions clear Did you find the instructions distributed for this PT adequate? Yes/No. If No, which parts do you think can be improved? 48 Answers
29 Comments Any other comments you wish to address? 26 Answers
42
Annex 7. Kernel density plots
The assigned (reference) values for DON and HT-2 toxin cluster very closely with the respective major modes and the robust means calculated from the results of the participants. The pairs of kernel density plots FB1/FB2 and HT-2/T-2 toxins show a similar and minor deviation from a Gaussian distribution. There is seemingly a significant number of laboratories which underestimated HT-2 and T-2 mass fractions, that despite using LC-MS/MS, didn't use 13C-labelled internal standards and the calibration standards were prepared in pure solvent. Although other participants following similar calibration strategy reached a satisfactory performance, this approach renders the procedure more vulnerable to systematic errors.
The deviation from normality in the AFB1 kernel density plot was investigated. Neither the different sample preparation techniques (IAC, dilute&shoot and QuEChERS) nor the analytical methods (LC-MS/MS and HPLC-FLD) used by or the participants could be unequivocally implicated in the apparent bimodality. Likewise, the origin of the calibration standards does not seem to have played any role in that regard.
Sample: CEREALS CORN, Measurand: DEOXYNIVALENOL
µg/kg1000900800700600500400300200
Pro
babi
lity
dens
ity
Low
er li
mit
of to
lera
nce
Upp
er li
mit
of to
lera
nce
Mean: 587.1 ± 32.6 µg/kg
Assigned value (Reference value): 611.0 ± 32.1 µg/kg
Mod
e 2:
268
.0 µ
g/kg
(5
%)
Mod
e 3:
593
.0 µ
g/kg
(92
%)
Sample: CEREALS CORN, Measurand: AFLATOXIN B1
µg/kg181614121086420
Pro
babi
lity
dens
ity
Low
er li
mit
of to
lera
nce
Upp
er li
mit
of to
lera
nce
Mean: 9.6 ± 0.6 µg/kg
Assigned value (Reference value): 10.6 ± 0.7 µg/kg
Mod
e 1:
3.7
µg/
kg (
5 %
)
Mod
e 2:
9.1
µg/
kg (
64 %
)
Mod
e 3:
11.
2 µg
/kg
(31
%)
43
Sample: CEREALS CORN, Measurand: FUMONISIN B1
µg/kg1400120010008006004002000
Pro
babi
lity
dens
ity
Low
er li
mit
of to
lera
nce
Upp
er li
mit
of to
lera
nce
Mean: 714.7 ± 60.2 µg/kg
Assigned value (Reference value): 767.8 ± 50.3 µg/kg
Mod
e 1:
660
.8 µ
g/kg
(93
%)
Mod
e 2:
122
6.4
µg/k
g (7
%)
Sample: CEREALS CORN, Measurand: FUMONISIN B2
µg/kg5004003002001000
Pro
babi
lity
dens
ity
Low
er li
mit
of to
lera
nce
Upp
er li
mit
of to
lera
nce
Mean: 196.1 ± 19.6 µg/kg
Assigned value (Reference value): 223.7 ± 16.4 µg/kg
Mod
e 1:
183
.2 µ
g/kg
(91
%)
Mod
e 2:
348
.2 µ
g/kg
(8
%)
Mod
e 3:
495
.1 µ
g/kg
(2
%)
Sample: CEREALS OAT, Measurand: HT-2
µg/kg4003002001000-100
Pro
babi
lity
dens
ity
Low
er li
mit
of to
lera
nce
Upp
er li
mit
of to
lera
nce
Mean: 145.4 ± 22.8 µg/kg
Assigned value (Reference value): 150.3 ± 9.5 µg/kg
Mod
e 1:
157
.5 µ
g/kg
(98
%)
Mod
e 2:
332
.0 µ
g/kg
(2
%)
Sample: CEREALS OAT, Measurand: T-2
µg/kg150100500
Pro
babi
lity
dens
ity
Low
er li
mit
of to
lera
nce
Upp
er li
mit
of to
lera
nce
Mean: 80.4 ± 7.8 µg/kg
Assigned value (Reference value): 70.3 ± 2.1 µg/kg
Mod
e 1:
78.
5 µg
/kg
(92
%)
Mod
e 2:
156
.6 µ
g/kg
(8
%)
44
Sample: CEREALS CORN, Measurand: ZEARALENONE
µg/kg250200150100500
Pro
babi
lity
dens
ity
Low
er li
mit
of to
lera
nce
Upp
er li
mit
of to
lera
nce
Mean: 151.4 ± 10.7 µg/kg
Assigned value (Reference value): 161.6 ± 8.8 µg/kg
Mod
e 1:
46.
8 µg
/kg
(5 %
)
Mod
e 2:
101
.4 µ
g/kg
(13
%)
Mod
e 3:
159
.3 µ
g/kg
(82
%)
Sample: CEREALS OAT, Measurand: ENNIATIN B
µg/kg806040200-20
Pro
babi
lity
dens
ity Mod
e 1:
37.
2 µg
/kg
(100
%)
Sample: CEREALS OAT, Measurand: ENNIATIN B1
µg/kg454035302520151050-5-10
Pro
babi
lity
dens
ity Mod
e 1:
18.
8 µg
/kg
(100
%)
45
Sample: CEREALS OAT, Measurand: ENNIATIN A1
µg/kg2520151050-5-10
Pro
babi
lity
dens
ity Mod
e 1:
5.3
µg/
kg (
79 %
)
Mod
e 2:
14.
1 µg
/kg
(21
%)
Sample: CEREALS OAT, Measurand: BEAUVERICIN
µg/kg6040200-20-40
Pro
babi
lity
dens
ity
Mod
e 1:
8.3
µg/
kg (
74 %
)
Mod
e 2:
31.
6 µg
/kg
(26
%)
46
Annex 8. Distribution of individual results and respective uncertainties (k=2)
Laboratory
LC00
17
LC00
43
LC00
20
LC00
56
LC00
29
LC00
21
LC00
45
LC00
14
LC00
40
LC00
46
LC00
30
LC00
26
LC00
16
LC00
19
LC00
22
LC00
41
LC00
36
LC00
51
LC00
33
LC00
15
LC00
54
LC00
42
LC00
48
LC00
31
LC00
38
LC00
24
LC00
52
LC00
09
LC00
44
LC00
18
LC00
01
LC00
49
LC00
12
LC00
27
LC00
34
LC00
39
LC00
28
LC00
50
LC00
35
LC00
25
LC00
37
LC00
55
LC00
02
LC00
32
LC00
07
LC00
11
LC00
05
LC00
47
µg/k
g
12001100
1000
900
800
700600
500
400
300200
100
Sample: CEREALS CORNMeasurand DEOXYNIVALENOLMethod: ISO 5725-5 (Alg. A+S)Number of laboratories in calculation: 48
Aflatoxin B1 - ISO 17375/2006 Fumonisin B1/B2 - EN 14352 and other DON, ZON, HT-2, T-2, ENN, BEAU several non-official
10 g and 100 ml acetonitrile/water - 84/16 shaking in 60 min, filtration, dissolving with acetonitrile/water - 84/16 and adding internal standard. Evaporation to dryness. Resolving in 1 ml 40% methanol.
Mutitoxin method Multi-toxin method "dilute and shoot", LC-MS/MS
Compilation of Waters LC/MS/MS application method and EURL method for multi mycotoxin determination in cereal based feed
weigh 5g sample, dilute with 25 mL of extraction solution (ACN/water/FA 79/20/1), vortex, shaking 1h, centrifuge, evaporation and redilution to MeOH/water 50/50
LC0006 Individual methods HPLC-FLD Sample 10 g Methanol/Water 20 ml (extraction)
LC0007 Corn and corn based products, dried fruits (pistachios and peanuts)
Mutitoxin method LC-MS/MS for all the tested mycotoxins
No official method used For T-2 and HT-2 the following method was applied: JRC 66507 EN - JRC - IRMM "Validation of an analytical method for the simultaneous determination of deoxynivalenol, zearalenone, T-2 and HT-2 toxins in unprocessed cereals - Validation report. Andreas Breidbach. 2011. The extraction of DON, AFB1, FB1, FB2 and ZON was performed by shaking for 60 minutes 2g of grounded sample with 8ml of AcCN:H2O:HCOOH 79:20:1
LC0008 Feed Materials Individual methods AflaB1-IAC-HPLC-FLD In House Method based on ISO 17375:2006 50g of sample, 250ml extraction solvent acetone/H2O (85/15), 30' shaking
LC0009 Corn, wheat Mutitoxin method extraction ASE, LC-MS/MS extraction with ASE (ACN, MeOH, water)
Mutitoxin method QuEChERS for all compounds In-house method 2-gram sample, extraction with acetonitrile/formic acid, addition of magnesium sulphate and sodium chloride, shaking, centrifugation, filtration through syringe filter, LC-MS/MS
LC0020 Individual methods AFLA - HPLC - FLD T2, HT2 - HPLC - FLD ZEA - HPLC - FLD DON - HPLC - DAD FUMO B1,B2 HPLC FLD
LC0021 cereal and cereal products Mutitoxin method dilute and shoot, LC-MS/MS no reference extract with ACN/water/acetic acid dilute 1:1 with ACN/water/acetic acid
LC0022 mixed feed Mutitoxin method LC-QQQ (dilute & shoot) ACN/water/acetic acid (89/20/1); 120 min stirring; 25g; 100 ml
LC0024 cereals (barley, oat, wheat and rye) feed (cereal based)
Mutitoxin method DON, T-2. TH-2, zearalenone, aflatoxin B1, fumonisins B1 and B2 - UHPLC-MS/MS enniatins and beauvericin - not analysed
EURL draft "Determination of deoxynivalenol, aflatoxin B1, fumonisins B1&B2, T-2 & HT-2 toxins, zearalenone and ochratoxin A in unprocessed cereals and cereal based compound feeds by liquid chromatography - tandem mass spectrometry.
Mutitoxin method LC/MSMS for all analytes analysed 1. The Detection of Mycotoxins Using a Simple Sample Extraction and LC-MS/MS with Fast Polarity Switching and the Scheduled MRM Algorithm. AB Sciex publication, Jianru Stahl-Zeng, Stephen Lock, Stefanie Krepperhofer, Kristen von Czapiewski 2. LC-MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs. Food additives and Contaminants, April 2008;25(4): 472-489, Martien C. Spanjer, Peter M. Rensen and Jos M. Scholten
80:20 acetonitrile:water
LC0029 peanuts, hazelnuts, pistachios, dried figs, red pepper, cereal crops such as wheat, corn, barley and wheat flour, corn oil.
AFL- EN ISO 16050 OTA EN 14132 DON EN 15891 ZEA - EN 15850 FUM -EN 14352
multitoxin IACs with one sample preparation, but different chromatographic methods and detectors wavelengths. AFL-OTA-FUM-IAC-HPLC_FLD- 10g sample with 2 g NaCl was extracted with 40 ml mixture of methanol-water=60/40. after filtration 10 ml were diluted with 15 ml PBS solution. After filtration with glass microfiber filter, 5 ml are passed through the IAC. The IAC column was washed with 20 ml water. eluting with 1 ml methanol with backflushing and 1 ml of water. DON-ZON_T2/HT2-IAC-HPLC_FLD_DAD -10g sample with 2 g NaCl was extracted with 40 ml mixture
AFLA IAC-HPLC-FLD FUMO IAC-HPLC-FLD DON-ZEA-T-2-HT-2 SPE-LC/MS/MS
In-house validated method based on draft for ongoing CEN mandate "Multimethod for the screening of ochratoxin A, aflatoxin B1, deoxynivalenol, zearalenone and fumonisin B1 and B2 in foodstuffs by LC-MS/MS"
DON-ZEA-T-2-HT-2 Extraction with Acetonitrile / water 84:16
LC0034 none (no routine analysis) Mutitoxin method All analytes: HPLC-MS/MS internal methods Samples were extracted with a mixture of 10 ml water and 10 ml acetonitrile containing 0.1% formic acid. After 30 min shaking and centrifugation, 1 ml of the supernatant was mixed with 100 µl isotope-labelled standard
51
solution and 250 mg MgSO4. After phase separation, 300 µl of the organic phase was mixed with 300 µl water and analysed using LC-MS/MS
LC0035 cereals, baby foods, dryed
fruits Individual methods IAC-LC-MS/MS for all mycotoxins Aflatoxins - EN 14123
T-2/HT-2 - Method of CRL 2006 Fumonisins - EN 14352 DON - EN 15891 ZON - EN 15850
AFLA: EN 15851:2010 DON: in-house method ZON: in-house method
Individual methods used
LC0043 feed animal tissues milk
Mutitoxin method LC-MS/MS Determination of Deoxynivalenol, Aflatoxin B1, Fumonisin B1&B2, T-2 & HT-2 toxins, Zearalenone and Ochratoxin A in unprocessed cereals and cereal-based compound feeds by Liquid Chromatography - Tandem Mass Spectrometry- DRAFT- SOP EURL GEEL
Determination of Deoxynivalenol, Aflatoxin B1, Fumonisin B1&B2, T-2 & HT-2 toxins, Zearalenone and Ochratoxin A in unprocessed cereals and cereal-based compound feeds by Liquid Chromatography – Tandem Mass Spectrometry- DRAFT- SOP EURL GEEL
LC0044 feed, raw materials and cereals for humans consumption
Aflatoxin B1 - EN ISO 17375 Fumonisins - EN 13585, CEN/TS 16187 DON - EN 15791 ZON - EN 15792 T-2, HT-2 - R-BIOPHARM RHONE LTD IAK Description
LC0050 food: dried fruits, cerelas and cereals products, nuts,spices,baby food feed: raw materials, compounds feed,pet food
Mutitoxin method/Individual methods
T2;HT2,DON,Zea: LCMS Fumos B1 and B2: IAC-HPLC- fluo after derivatization (OPA) AfB1: IAC-HPLC-fluo after derivatization with Cobra cell
Afla B1: NF EN 14123 Fumos B1 and B2 : NF EN 16006 T2 HT DON ZEA: in-house method lcms
for Don,THT2,DON and Zea: - Extraction : Weigh 10g of sample ( 1mg precision)in an erlen, add 100ml of extraction solution : CH3CN/H2O/CH3COOH - 80/20/1 Agitate magnétically during 1h Filtration with paper - Preparation of extract injection : Transfer 1ml (=0,1g) of extract in a vial deactivated of 4ml. Add 25 µl of Mix IS in all vials at the same time (standards solutions and samples) with multipet and evaporate dry with nitrogen. Solubilize the residue in 500µl of mobile phase containing 1mM ammonium acetate + 0.1% acetic acid as follow: - Add 100µl of mobile phase B (M"
LC0051 Cereals Mutitoxin method MULTI IAC - UHPLC - HRMS/MS Double extraction: PBS and methanol
LC0053 cereals, coffee, dried fruit, spices, wine, apple based products, beer
Individual methods Aflatoxins B1- HPLC-FL UNI EN ISO 16050 2011 methanol/water 80/20 extraction
LC0054 Cereals Mutitoxin method Aflatoxin B1 - IAC-HPLC-FL The other mycotoxins - UHPLC-MS/MS
In-house methods AcN - acetonitrile/water 80/20 (v/v) The other mycotoxins - acetonitrile/water/formic acid 74/25/1 (v/v/v)
LC0055 Cereals, nuts, baby food, milk, apple juice, wine.
Mutitoxin method UPLC-MS/MS Multitoxin method House Method based in the article Determination of mycotoxins in different food commodities by UPLC-MS/MS. Rapid Commun. Mass Spectrum. 2009; 23; 1801-1809.
10 g + 40 ml Acetonitrile 80% + 0.1% formic acid. Shaking 90 min. Centrifuge. Dilute 1 ml extract + 1 ml H2O. Filter 0.2 um DON : 1 ml extract, evaporate (dryness). Add 1ml H2O. Filter 0.2 um.
LC0006 Vicam Aflatest T-2 489.6>327.1; HT-2 489.6>345.1; DON 297.3>231; AFB1 313>285; FB1 723.3>335.1; FB2 706.9>336.5; ZON 319>187. ESI+ for all the texted mycotoxins
Standard in pure solvent
LC0007 No IAC or SPE used for clean-up the samples.
isotope-labelled standard was used for the determination of T-2, HT-2; DON, AFB1, FBs
For T-2, HT-2, DON, AFB1, FB1, FB2 standards in pure solvent with the addition of the isotope-labelled standard was used. For ZON matrix, matched calibration was used.
LC0008 LC-Tech IAC Standards in pure solvent LC0009 FB1: ES+ 722.4>352.4 / FB2: ES+ 706.5>336.4/ AF B1:
yes, for all toxins except enniatins and beauvericin
After extraction
Standards in pure solvent
57
Enn A1 - ESI+ m/z 686>210 Enn B - ESI+ m/z 658>196 Enn B1 - ESI+ m/z 672>196 Beau - ESI+ m/z 802>244
LC0041 NA AFB1 - ESI+ m/z 313>285 NA Standards in pure solvent. LC0042 AFLA, DON, ZON: r-Biopharm Solvents in pure solvent LC0043 n.a. DON - ESI NEG 355>265.1
ZEN - ESI NEG 317>131 AFLB1 ESI POS 313>241 FB1 ESI POS 722.2>352.2 FB2 ESI POS 706.5>336.6 BEA ESI POS 801>134 ENN A ESI POS 699.3>210.2 ENN A1 ESI POS 685>210 ENN B ESI POS 657>196 ENN B1 ESI POS 671>196 T-2 ESI POS489>245 HT-2 ESI POS 442>215
LC0001 From initial method validation data/ Long term compilation of quality control data
Spiking/ Certified Reference Material
Corrected for recoveries No. We did earlier, but the quality is too bad. We have our own in-house material and are participating in many PT´s
DON - Biopure, ZON - Biopure, HT-2 - Biopure, T-2 - Biopure, ENN A - Sigma/Aldrich, ENN A1 - Sigma/Aldrich, ENN B - Sigma/Aldrich, ENN B1 - Sigma/Aldrich, BEAU - Sigma/Aldrich
Protection from daylight, especially for aflatoxins
LC0002 From initial method validation data
Spiking Not corrected for recoveries
No FLUKA NaOCl -washing of the glassware, amber glassware and protection from daylight
LC0004 Long term compilation of quality control data
For all mycotoxins tested we use ambar glassware and protection from daylight
LC0005 Other Spiking Corrected for recoveries No No LC0006 From initial method
validation data Spiking Corrected for recoveries Sigma Aldrich Aflatoxin
B1+B2+G1+G2
LC0007 Other Spiking Corrected for recoveries No Biopure, for all the tested mycotoxins
No special precautions were taken, but no new glassware was used. Sample extracts were protected from daylight by wrapping the tubes with aluminium foil
LC0008 Long term compilation of quality control data
Spiking No SIGMA Yes for aflatoxin B1
LC0009 From initial method Spiking/ Certified Not corrected for PT Bipea (corn and oat) Romer Labs
60
validation data Reference Material recoveries LC0011 Long term compilation of
quality control data Spiking No R-Biopharm Protection from daylight
LC0012 From initial method validation data
Spiking Corrected for recoveries R-BIOPHARM (TRILOGY) AMBAR GLASSWARE
LC0013 Long term compilation of quality control data
Spiking Corrected for recoveries No B1: Biopure, Romer Labs
LC0014 From initial method validation data
Spiking - No
LC0015 From initial method validation data
Spiking Corrected for recoveries No DON, ZON, AFB1, FB1, FB2, T2, HT2: Sigma Aldrich BEA, ENN A, ENN A1, ENN B, ENN B1: Enzo Lifesciences
amber vials, acid washing glassware
LC0016 From initial method validation data
Spiking/ Certified Reference Material
Corrected for recoveries TRILOGY NO SPECIAL PRECAUTIONS
LC0017 Long term compilation of quality control data
Spiking Corrected for recoveries Acid-washed glassware
LC0018 Other Spiking Corrected for recoveries Not yet regularly several no glassware, only disposables
LC0019 From initial method validation data
Spiking Corrected for recoveries LGC Standards, protection from daylight
LC0020 From initial method validation data
Spiking/ Certified Reference Material
Not corrected for recoveries
LC0021 No sigma aldrich No LC0022 From initial method
validation data Spiking Not corrected for
recoveries No Romer Labs No
LC0024 From initial method validation data
Spiking/ Other Not corrected for recoveries
not yet Sigma, Biopure acid washing of the glassware deactivated vials protection from the sunlight
LC0025 Long term compilation of quality control data/ Other
Spiking/ Other Corrected for recoveries Just Reference Materials from FAPAS for DON,ZON,FB1FB2 - Maize flour
SIGMA acid washing of the glassware, use of amber glassware, precaution during the evaporation step (ZON,DON,FB1FB2) and protection from daylight
LC0026 From initial method validation data
Not corrected for recoveries
No all mycotoxins are protected from daylight by using special glassware
LC0027 Long term compilation of quality control data
Romer Labs (Biopure) deactivated glassware; dark vials
LC0028 From initial method validation data
Certified Reference Material
Not corrected for recoveries
FAPAS sample analysed in every batch, maize, Zon, Don, T-2. HT_2, AFB1, OTA, Fum B1, Fum B2
LGC We use silanised glassware for fumonisin stds
LC0029 From initial method validation data
Spiking Not corrected for recoveries
We don't use CRM; we use QC materials of FAPAS in matrices like nutmeg,dried figs, pistachio, red pepper
Biopure
Yes for aflatoxins -acid washing of the glassware and amber glassware
LC0030 From initial method validation data/ Long term
Corrected for recoveries No SIGMA ALDRICH No
61
compilation of quality control data
LC0031 From initial method validation data
Spiking/ Certified Reference Material
Corrected for recoveries Corn naturally contaminated with mycotoxins (aflatoxins, DON, OTA, T-2, HT-2, ZON, FB1 and FB2): Trilogy Analytical laboratory
Sigma Aldrich (aflatoxins, DON, OTA, T-2, HT-2, ZON, FB1 and FB2), Enzo life sciences (Enns + Bea)
Aflatoxins: protection from daylight
LC0032 Other Spiking Corrected for recoveries - T-2, HT-2 - Romer Fumonisin - Romer DON - Romer Aflatoxin - Supelco F2 - Romer
standards and samples are protected from daylight
LC0033 From initial method validation data
Spiking Corrected for recoveries DON-ZEA-T-2-HT-2 Maize Flour by FAPAS Orsell No
LC0034 Other Spiking/ Certified Reference Material
Corrected for recoveries Aflatoxin B1, Deoxynivalenol, Zearalenone: maize quality-control test material supplied by FAPAS (T04201QC) T2-Toxin, HT2-Toxin: oat flakes certified reference material supplied by BAM (ERM-BC720) Fumonisin B1: maize powder from 2013 EURL-PT multitoxin
Aflatoxin B1: IRMM (ERM AC057) certified reference standard solution Deoxynivalenol, Fumonisin B1+B2; HT2-Toxin, T2-Toxin, Zearalenone: Romer Labs "Biopure" certified standard solutions Enniatins, Beauvericin: Cfm Oskar Tropitzsch, bulk
amber, silanised glassware, including HPLC vials
LC0035 Other Spiking Corrected for recoveries No Biopure No LC0036 Long term compilation of
quality control data Spiking Sigma-Aldrich amber glassware
protection from daylight LC0037 From initial method
validation data Spiking Corrected for recoveries No Romerlabs Aflatoxins
LC0038 From initial method validation data
Spiking Not corrected for recoveries
No Romer Labs, biopure No
LC0039 Long term compilation of quality control data
Spiking Corrected for recoveries FAPAS animal feed;, DON, T2, HT2 and Zearalenone
Sigma-Aldrich Biopure
No
LC0040 From initial method validation data/ Long term compilation of quality control data
Spiking Corrected for recoveries No Romer Labs Acid washing of glassware, amber LC-vials, protection from UV (window filters and LED light)
LC0041 Other Spiking Corrected for recoveries NA No. LC0042 Long term compilation of
quality control data/ Other Spiking Corrected for recoveries CRMs are sometimes used for Aflatoxin
analysis for nuts and dried fruit matrices supplied by FAPAS
AFLA: Sigma DON: Biopure ZON: Biopure
Amber glassware and protection from daylight are applied through analysis
LC0043 Other Spiking Corrected for recoveries Yes, but not for this analysis. Sigma Aldrich Romer labs
polypropylene tubes, amber vials
LC0044 Long term compilation of quality control data
Spiking Corrected for recoveries Yes, we use for quality control, and the supplier is R- Biopharm/Trilogy
SUPELCO/ALDRICH Acid washing of glassware, amber glassware and protection from daylight
LC0045 From initial method validation data
Spiking Corrected for recoveries No AFLA - Sigma Aldrich LGC for all others
-
LC0046 Long term compilation of quality control data/ Other
Spiking Not corrected for recoveries
No Biopure except for Enniatins et beauvericin (Sigma-Aldrich)
acid washing of the glass ware
LC0047 Long term compilation of quality control data
Spiking Corrected for recoveries No Romerlabs No
62
LC0048 From initial method validation data/ Long term compilation of quality control data
Certified Reference Material
Corrected for recoveries No Sigma Daylight protection
LC0049 Long term compilation of quality control data
Spiking Corrected for recoveries FAPAS, various matrices Aflatoxin B1 - SUPELCO Fumonisins - SIGMA-ALDRICH DON - BIOPURE ZON - BIOPURE T-2, HT-2 - BIOPURE
For Aflatoxin B1 - acid washing of the glassware, for all mycotoxins - amber glassware and protection from daylight.
LC0050 Long term compilation of quality control data
Spiking Corrected for recoveries No DON: Libios Zéa:Libios AflaB1: libios T2 and HT2 and fumos B1 and B2: Biopure
acid washing of the glassware and protection from daylight
LC0051 Corrected for recoveries No Romer No LC0052 From initial method
validation data Spiking Corrected for recoveries Zearalenone: ERM Wheat
DON: ERM Wheat Biopure, Supelco
LC0053 From initial method validation data
Spiking Corrected for recoveries We use CRMs for aflatoxins. Romer Labs Protection from daylight
LC0054 From initial method validation data
Spiking Corrected for recoveries Yes. Fapas QC materials Aflatoxin B1 - Sigma Aldrich The other mycotoxins - Biopure
Acid washing of glass ware (all mycotoxins), daylight protection (aflatoxin B1)
LC0055 From initial method validation data
Spiking No Sigma Aldrich Ambar glassware and protection from daylight.
LC0056 Other Spiking Corrected for recoveries No Romer Labs, BioPure Silanised UPLC Vials
Rec – recovery (%), LOD – limit of detection (µg kg-1), LOQ - limit of quantification (µg kg-1)
65
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